The current nucleic acid sequencing methods are able to read millions of individual sequences. These next-generation sequencing (NGS) technologies also known as massively parallel sequencing (MPS) utilize amplified nucleic acids derived from the original sample. Amplification error and bias inherent to PCR impair accuracy of sequence reads and any quantitative analysis of the sequences in the sample. These errors compromise clinical utility of the sequencing data. Typical NGS platforms need a high level of redundancy “sequence coverage” to overcome the limitation resulting from such errors. PCR amplification biases and errors are especially prominent in a multiplex reaction, such as library preparation. There is an unmet need for a method of amplifying multiple templates without bias and with a low rate of error.
PCR Amplification Bias
PCR is the most widely used method to generate large quantities of target nucleic acid amplicons, but it is understood that PCR is prone to amplification biases especially in the case of PCR multiplexing. These amplification biases are typically caused by inescapable differences in primer melting temperatures (Tm) from target to target but understood to also be caused by target insert secondary structure, GC content and length. Due to the exponential nature of PCR, each inefficiency of product extension per cycle due to priming or insert content can lead to large differences in final yield of each amplicon comparatively. For example, given templates with various known doubling efficiencies per cycle, one could predict the relative final yields of these products following differing numbers of PCR cycles. As can be seen in Table 1 below, after 10 cycles of PCR amplification, the relative yield of the 99% efficiency/cycle product would be expected to be 2.5 fold higher than the 90% efficiency/cycle product (90%/35%). Larger differences in product extension efficiencies and/or increasing the number of cycles will lead to even larger difference in yield of each product.
TABLE 1Hypothetical product yield at various PCR efficienciesCycle99% eff.95% eff.90% eff.199%95%90%298%90%81%397%86%73%496%81%66%595%77%59%694%74%53%793%70%48%892%66%43%991%63%39%1090%60%35%
PCR Error Accumulation
PCR is also inherently error-prone. When polymerase errors occur in PCR, these errors are propagated to products generated in later cycles. For example, when targeting a 1 kb amplicon and using a polymerase that incorporates one error per 10,000 bases, 10% of newly generated products will contain a newly added error and only 90% of the new products will be error free. In the first round of cycling, 95% of the total molecules (template—100% and product—90%) would be error free. As can be seen in Table 2 below, when cycle number increases, the relative amount of error free products decreases.
TABLE 2Accumulation of errors during PCR cyclesError freeTotalPercent ErrorCyclemoleculesmoleculesFree molecules0100100100% 119020095%236140090%368680086%41,3031,60081%52,4763,20077%64,7056,40074%78,93912,80070%816,98425,60066%932,26951,20063%1061,311102,40060%11116,490204,80057%12221,331409,60054%13420,530819,20051%14799,0071,638,40049%151,518,1133,276,80046%162,884,4146,553,60044%175,480,38713,107,20042%1810,412,73526,214,40040%1919,784,19752,428,80038%2037,589,973104,857,60036%